The present invention relates to methods for designing 2,5-diketo-D-gluconic acid reductase enzymes with altered NADPH and NADH cofactor dependency by site-directed mutagenesis of one or more amino acids in the cofactor specificity site of the 2,5-diketo-D-gluconic acid reductase enzyme. The present invention also relates to mutated forms of the enzyme having altered NADPH and NADH cofactor dependency, DNA encoding these mutant enzymes, and vectors and host cells expressing these mutant enzymes. Mutated forms of the present invention exhibit non-wild type enzymatic activity with NADH, non-wild type activity with NADPH, activity with both NADPH and NADH, and altered expression characteristics.
L-ascorbic acid, also known as vitamin C, is an essential part of the diet and an important antioxidant. Increasing health consciousness of the public and use of this product in preservation of food has led to an increased demand for vitamin C. It is estimated that world-wide consumption of this specialty chemical exceeds 50,000 tons/year.
Vitamin C is industrially produced by the modified Reichstein and Grussner synthesis which requires one fermentation step by Acetobacter suboxydans and five chemical steps. A dual fermentation system has also been described wherein D-glucose is converted to 2,5-diketo-D-gluconic acid by Erwinia herbicola. The 2,5-diketo-D-gluconic acid is then converted to 2-keto-L-gulonic acid, a direct precursor of L-ascorbic acid, by Corynebacterium sp. (Sonoyama et al. App. Environ. Microbiol. 1982 43:1064-1069). This precursor is converted to ascorbic acid through acid or base catalyzed cyclization. Since this intermediate has greater shelf stability and shelf life than ascorbic acid, it is more practical to stockpile 2-keto-L-gulonic acid for subsequent conversion to vitamin C.
Production of 2-keto-L-gulonic acid from D-glucose via a single fermentation step has been described by Anderson et al. (Science 1985 230:144-149). In this process, 2,5-diketo-D-gluconic acid reductase (2,5-diketo-D-gluconic acid reductase A, also known as 2,5-diketo-D-gluconic acid reductase II) was cloned from Corynebacterium sp. and expressed in Erwinia herbicola. The resulting metabolically-engineered organism can produce 2-keto-L-gulonic acid from D-glucose in a single fermentation step.
A method for producing 2-keto-L-gulonic acid from glucose in vitro was also recently described (Boston et al. Biotrans. 1999: 4th International Symposium on Biocatalysis and Biotransformation).
While high yields of 2-keto-L-gulonic acid are obtained with Corynebacterium sp. 2,5-diketo-D-gluconic acid reductase, the compound 2,5-diketo-D-gluconic acid is actually a poor substrate for this enzyme. Accordingly, various attempts have been made to improve the catalytic efficiency, thermal stability and/or expression levels of 2,5-diketo-D-gluconic acid reductase in this single fermentation process.
U.S. Pat. Nos. 5,376,544, 5,583,025, 5,912,161 and 5,795,761 disclose mutants of 2,5-diketo-D-gluconic acid reductase A with increased catalytic activity, increased expression levels, and/or enhanced temperature stability. Mutants disclosed in U.S. Pat. Nos. 5,376,544, 5,583,025, and 5,912,161 include replacement or deletion of the amino acid residue at position 2, 5, 7, 55, 57, 165, 166, 167, 168, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277 or 278 of wild type 2,5-diketo-D-gluconic acid reductase A. Mutants disclosed in U.S. Pat. No.5,795,761 include replacement or deletion of the amino acid residue at position 21, 22, 23, 24, 25, 46, 47, 48, 49, 50, 51, 52, 164, 169, 170, 199, 200 or 235 of wild type 2,5-diketo-D-gluconic acid reductase A.
The 2,5-diketo-D-gluconic acid reductase enzyme exhibits a dramatic preference for NADPH over NADH. When the available concentration of NADPH is low, then overall activity of the enzyme is limited. However, increasing the available concentration of NADPH in the cell can be difficult and increasing the amount added to an in vitro reaction can be quite expensive as NADPH is a costly component.
The crystal structure of wild type 2,5-diketo-D-gluconic acid reductase A with bound NADPH has been disclosed (Khurana et al. Proc. Natl Acad. Sci. 1998 95:6768-6773). Sequence alignment with the NADPH cofactor binding loop of similar enzymes such as aldose reductase has also been performed (Rondeau et al. Nature 1992 355:469; Wilson et al. Science 1992 257:81; Jez et al. Biochem. J. 1997 326:625-636). Accordingly, some of the residues of this enzyme that interact with the NADPH molecule have been delineated.
A poster was presented by Banta et al. on Nov. 16, 1998 at the Annual Meeting of the American Institute of Chemical Engineers suggesting improving the NADPH kinetics of the enzyme as a means for improving the overall catalysis. However, no means for achieving this were described. Nor was there any mention of improving NADH kinetics.
An object of the present invention is to provide a method of producing mutant 2,5-diketo-D-gluconic acid reductase enzymes with altered cofactor dependency, which comprises identifying a cofactor specificity site in the enzyme and mutating an amino acid in the identified cofactor specificity site of wild type 2,5-diketo-D-gluconic acid reductase.
Another object of the present invention is to provide mutant 2,5-diketo-D-gluconic acid reductase enzymes with altered cofactor dependency, DNA encoding these mutant enzymes, and vectors and host cells expressing these mutant enzymes.